It is known that there are simple nucleotide sequences in the human genome that can occur in different numbers of repeats in different individuals, giving rise to a range of different alleles or variants of different length that can be used as genetic markers to typify the DNA of an individual. It is these genetic markers that DNA diagnostic laboratories use in molecular diagnostics procedures for the identification and characterization of diseased genes. Such genetic markers are also use for precision of DNA typing of individuals in the field of forensic science.
In general, tandem repeat minisatellite and microsatellite regions in vertebrate DNA frequently show high levels of allelic variability in the number of repeat units. These highly informative genetic markers have found widespread applications in population genetics, forensic science, medicine and other natural scientific studies. For example, these markers can be used for linkage analysis, determination of kinship in paternity and immigration disputes and for individual identification in forensic medicine. In a minisatellite system, a core DNA sequence unit is usually 15 or more base pairs. To date most studies and applications of such systems have relied on Southern blot estimation of allele length, which requires at least 50 ng of relatively undegraded DNA. It is often very difficult to extract such large amounts of DNA from many forensic samples such as blood and semen stains.
In recent years, the discovery and development of polymorphic short tandem repeats (STRs) and Variable Number Tandem Repeats (VNTRs) as genetic markers have stimulated progress in the development of linkage maps, the identification and characterization of diseased genes, and the simplification and precision of DNA typing of individuals.
Many loci in the human genome contain a polymorphic STR region. STR loci consist of short, repetitive sequence elements on the order of 3 to 7 base pairs in length. It is estimated that there are roughly 2,000,000 expected trimeric and tetrameric STRs present as frequently as once every 15 kilobases (kb) in the human genome (Edwards et al. 1991 (Am J Hum Genet 49:746-756); Beckmann and Weber 1992 (Genomics 12:627-631)). Nearly half of the STR loci studied by Edwards et al. (1991) are polymorphic, which provides a rich source of genetic markers. Variation in the number of repeat units at a particular locus is responsible for the observed polymorphism reminiscent of VNTR loci (Nakamura et al. 1987 (Science 235:1616-1622) and minisatellite loci (Jeffreys et al. 1985 (Nature 316:76-79)), which contain longer repeat units, and microsatellite or dinucleotide repeat loci (Litt and Luty 1989 (Am J Hum Genet 44:397-401), Tautz 1989 (Nucleic Acids Res. 17:6463-6471), Weber and May 1989 (Am J Hum Genet 44:388-396), Beckmann and Weber 1992 (Genomics 12:627-631)).
Such polymorphic STR loci are extremely useful markers for human identification, paternity testing and genetic mapping. STR loci may be amplified via the polymerase chain reaction (PCR) by employing specific primer sequences identified in the regions flanking the tandem repeat. Alleles of these loci can be differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another following electrophoretic separation by any suitable detection method including, for example, radioactivity, fluorescence, silver stain, and color. To minimize labor, materials and analysis time, it is desirable to analyze multiple loci and/or more samples simultaneously. One approach involves amplification of multiple loci simultaneously in a single reaction. Such “multiplex” amplifications have been described extensively in the literature, for example, in the analysis or genes related to human genetic diseases such as Duchenne Muscular Dystrophy (Chamberlain et al. 1988 (Nucleic Acid Res 16: 11141-11156), Chamberlain et al. 1989 (“Multiple PCR for the diagnosis of Duchenne muscular dystrophy,” In PCR Protocols, A Guide to Methods and Application (ed. Gelfand, D. H., et al.) pp. 272-281. Academic Press, San Diego, Calif.), Beggs et al. 1990 (Hum. Genet. 86: 45-48), Clemens et al. 1991 (Am J. Hum. Genet. 49: 951-960), Schwartz et al. 1992 (Am J. Hum. Genet. 51: 721-729), Covone et al. 1992 (Am. J. Hum. Genet. 51: 675-677)), Lesch-Nyhan Syndrome (Gibbs et al. 1990), Cystic Fibrosis (Estivill et al. 1991 (Lancet 338: 458), Fortina et al. 1992 (Hum Genet. 90: 375-378), Ferrie et al. 1992 (Am. J. Hum. Genet. 51: 251-262), Morral and Estivill, 1992 (Genomics 51:1362-1364), and Retinoblasma (Lohmann et al. 1992 (Hum. Genet. 89: 49-53)). Multiplex amplification of polymorphic microsatellite markers and STR markers have been described previously in the literature (Clemens et al. 1991 (Am J. Hum. Genet. 49: 951-960), Schwartz et al. 1992, Huang et al. 1992 (Genomics 13: 375-380), Edwards et al. 1992 (Genomics 12:241-253), Kimpton et al. 1993 (PCR Methods and Applications 3: 13-22), Hammond et al. 1994 (Am. J. Hum. Genet. 55: 175-189)).
Recently, RFLPs that have Variable Number Tandem Repeats (VNTRs) have become a method of choice for human mapping because such VNTRs tend to have multiple alleles and are genetically informative because polymorphisms are more likely to be segregating within a family. The production of fingerprints by Southern blotting with VNTRs (Jeffreys et al., Nature 316:76-79 (1985)) has proven useful in the field of forensics. There are two classes of VNTRs; one having repeat units of 9 to 40 base pairs, and the other consisting of minisatellite DNA with repeats of two or three base pairs. The longer VNTRs have tended to be in the proterminal regions of autosomes. VNTR consensus sequences may be also used to display a DNA fingerprint.
Thus, while molecular diagnostics procedures, which rely on the use of such markers as, inter alia, STRs and VNTRs, are particularly well-suited to the application of prenatal diagnosis, presymptomatic diagnosis, carrier detection, and genetic screening, there still remain major bottlenecks in molecular diagnostic laboratories including front-end tasks such as sample purification and reaction setup. To date, the major sources for concern in clinical molecular laboratories are the safety, costs and efficiency of the normal procedures for preparation of specimens, such as blood, prior to analysis. Blood specimens for clinical analyses are commonly collected in evacuated blood collection tubes. Serum or plasma may be isolated from the cellular material by centrifugation and transferred to one or more specialized sample vessels. These sample vessels are used to introduce a portion of the specimen to chemical analyzers. However, the large numbers of samples involved often presents significant problems with sample tracking and data exchange between different laboratory instruments and information management systems. A certified DNA Diagnostics Laboratory provides a chain of custody report for each sample that is to be tested. The report traces the history of each sample from the time it was collected by one of their representatives until the results are released. The DNA Diagnostics Laboratory usually relies on a computerized sample tracking system that assigns a number for each sample to ensure confidentiality and chain of custody.
To provide for proper biological sample identification, a computer readable bar code label is usually affixed to the tube containing the biological sample. The bar code label allows for electronic processing of the sample and also helps to eliminate misidentification or confusion of samples. While the use of computer-based barcodes can provide a high level of sample tracking, such barcode labels still suffer from some significant drawbacks. For example, they are susceptible to manipulation, they typically involve an additional step, they can be lost, and barcodes are not unique to the individual, etc. In addition, the time and technical constraints associated with most sample preparation protocols have heretofore impeded the rapid, cost-effective, reproducible, systematic and unequivocal identification of biological samples.
For these aforementioned reasons, what is needed in the field of diagnostic medicine and disease diagnosis is a system and method suitable for biological sample tracking without the prior possibilities of accidental misidentification of the source of the sample and any diagnostic data derived from such a sample. This application addresses these and other needs by providing a method for analyzing a biological sample to detect the presence of an infectious agent, a disease condition and/or disease predisposition while simultaneously determining the molecular barcode of the sample so as to uniquely identify the biological sample without the chance of any mishandling or misidentification. The invention also includes a microfluidic processor apparatus for use in such a method.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.